Glutamine: fructose-6-phosphate aminotransferase 2 (GFPT2) is a novel NF-B target that links cancer metabolism with metastatic phenotypes in non-small cell lung cancer Szymon Jakub Szymura Krakow, Poland M.Sc. Biotechnology, Jagiellonian University, 2010 M.Sc. Biochemistry, University of Virginia, 2012 A Dissertation Presented to the Graduate Faculty of the University of Virginia in Candidacy for the Degree of Doctor of Philosophy Department of Biochemistry and Molecular Genetics University of Virginia May 2016 ____________________________________ ____________________________________ ____________________________________ ____________________________________ i ABSTRACT Lung cancer is one of the most prevalent cancer types and the leading cause of cancer related mortality in the world. Non-small cell lung cancer (NSCLC) constitutes the majority of lung cancers with the five-year survival rate at 17%. Poor survival rates are due to late-stage diagnosis and limited response to standard of care cisplatin-based chemotherapy. Recent efforts in targeted therapy in lung cancer yielded limited success due to the activation of compensatory survival mechanisms and acquisition of drug resistance. Metastasis in NSCLC is driven by the trans-differentiation process called epithelial to mesenchymal transition (EMT) that promotes migratory and invasive phenotypes in cancer cells. EMT is induced by cytokines secreted by cells present in tumor microenvironment. The Mayo laboratory has shown that transforming growth factor beta (TGF) and tumor necrosis factor (TNF) cooperate to induce EMT in NSCLC and that activation of the nuclear factor kappa B (NF-B) pathway is essential for the transition. Cancer cells show elevated uptake and utilization of glucose and glutamine via aerobic glycolysis (Warburg effect) and anaplerotic TCA cycle respectively. Glucose and glutamine are both required for the synthesis of UDP-N-acetylglucosamine (UDP-GlcNAc) in the hexosamine biosynthesis pathway (HBP). UDP-GlcNAc is a precursor molecule for multiple processes including protein glycosylation, generation of glycosaminoglycans and O-GlcNAcylation of nucleocytoplasmic proteins. Aberrant elevation of glycans has been implicated in cancer progression, survival ii and metastasis. Glutamine: fructose-6-phosphate (GFPT) is a first and rate- limiting enzyme in HBP. Here we show that neuronal-specific GFPT2 isoform is a novel NF-B target, up-regulated during TNF/TGF-induced EMT in NSCLC and in response to MEK inhibition in KRAS-mutant NSCLC. GFPT2 induction elevates protein O-GlcNAcylation in mesenchymal cells and is required for the migration and invasion of mesenchymal NSCLC. Consistent with these observations high GFPT2 expression co-relates with poor clinical outcome of lung adenocarcinoma patients. iii ACKNOWLEDGEMENTS I would like to thank Dr. Marty Mayo for accepting me to his laboratory, helping me with my application to the UVA graduate school and being my mentor for the past years. I would like to thank him for his tremendous support and patience thorough my time in graduate school and for teaching me a great amount about cancer biology. I would like to thank Dr. Joel Hockensmith for accepting me into BMG program. I would like to thank Dr. Zygmunt Derewenda for leading a Polish student exchange program that gave me and others invaluable opportunity to study at the University of Virginia. I would like to thank my committee members: Dr. Stefan Bekiranov, Dr. David Kashatus and Dr. Patrick Grant for their constructive suggestions and encouragement. I would like to thank my former mentors Dr. Joanna Cichy and Dr. Derk Amsen for introducing me to the world of science and for being truly inspiring role models. I would like to thank Dr. David Allison for being my friend and mentor during my first years in the U.S. as well as Jackie D’Innocenzi for being a great friend, an inspiring human being and a brilliant labmate. I would also like to thank Jacob Zaemes for his great help and all present and former Mayo lab members including Sheena Clift, Lisa Gray, Brian McKenna, Jake Wamsley, Emily Glidden and others for their collaboration and help. Finally I would also like to thank Debbie Sites for her patience and help with all formalities of the graduate school. I would like to especially thank Ewelina Zasadzińska, and my family and friends for their support and understanding. iv TABLE OF CONTENTS Abstract i Acknowledgements iii Table of Contents iv List of Figures vii List of Abbreviations ix Chapter I: General Introduction 1 Lung Cancer 2 Metastasis and Epithelial to Mesenchymal Transition 7 Cancer Metabolism 12 Hexosamine Biosynthesis Pathway 19 Proteoglycans and Hyaluronan in Cancer Biology 24 N- and O-Glycosylation and Cancer Biology 29 O-GlcNAcylation in Cancer Biology 35 NF-B Pathway and Cancer 41 References 48 Chapter II: Materials and Methods 71 Cell Culture and Reagents 72 Plasmids and Cell Transfection 72 Viral Particle Production 73 Generation of Stable Cell Lines 74 Tumorsphere Cultures 75 ChIP-seq Data Analysis 76 Quantitative Real-Time Polymerase Chain Reaction v (QRT-PCR) and PCR 76 Chromatin Immunoprecipitation (ChIP) 76 Adenoviral Infections 77 Transwell migration and invasion assays and wound healing assays 78 Immunoprecipitation, succinylated Wheat Germ Agglutinin (sWGA) pull-down and Immunoblotting 79 Luciferase Reporter Assay 79 References 81 Chapter III: NF-B Upregulates Glutamine-Fructose-6-Phosphate Transaminase 2 (GFPT2) to Promote Migration in Non-Small Cell Lung Cancer 82 Abstract 84 Introduction 85 Results 88 OGT is required for EMT in A549 cells 88 Mesenchymal NSCLC cells upregulate genes involved in UDP-GlcNAc synthesis 91 Elevated GFPT2 expression correlates with poor clinical outcome in NSCLC 95 GFPT2 is an immediate-early gene product maintained in mesenchymal NSCLC cells 100 The GFPT2 gene is transcriptionally regulated by NF-B 104 GFPT2 regulates protein O-GlcNAcylation and migration of mesenchymal NSCLC cells 108 Discussion 112 References 118 Supplementary Figures 124 vi Chapter IV: NF-B, GFPT2 and hexosamine biosynthesis pathway are induced in response to MEK inhibition in KRAS-mutant Non-Small Cell Lung Cancer to promote chemoresistance 137 Abstract 138 Introduction 139 Results 142 MEK inhibition activates Akt and NF-B pathways in KRAS-mutant NSCLC cells 142 Inhibition of NF-B promotes apoptosis following MEK inhibition in KRAS-mutant LUAD cells 143 MEK inhibition induces protein O-GlcNAcylation and the expression of the GFPT2 gene 146 GFPT2 elevates protein O-GlcNAcylation and sustains NF-B signaling 152 Silencing of GFPT2 promotes cell death following MEK inhibition in KRAS-mutant LUAD cells 155 Discussion 159 References 162 Chapter V: Summary and Future Directions 166 Summary 167 Future Directions 169 Determine the mechanism of GFPT2-dependent effect on the migration of mesenchymal NSCLC cells 169 Determine the relevance of the GFPT2 induction in mesenchymal NSCLC cells in vivo 177 Determine the role of NF-B, GFPT2 and protein O-GlcNAcylation in the survival of KRAS-mutant NSCLC cells following MEK inhibition 182 References 189 vii LIST OF FIGURES Introduction Figure 1: Metabolic pathways of glucose and glutamine in a cancer cell 15 Table 1: Primer Sequences 82 Figure 1: OGT is required for epithelial to mesenchymal transition in NSCLC 89 Figure 2: O-GlcNAcylation and enzymes involved in the synthesis of UDP-GlcNAc are elevated in mesenchymal NSCLC 92 Figure 3: GFPT2 is induced in mesenchymal NSCLC cells and is co-expressed with mesenchymal markers in LUAD patients 96 Figure 4: GFPT2 is an immediate-early gene induced by TNF and maintained in mesenchymal NSCLC 101 Figure 5: GFPT2 is a direct target of NF-B 105 Figure 6: GFPT2 regulates migration of mesenchymal NSCLC 109 Supplementary Figure S1: GFPT1 expression in mesenchymal NSCLC 125 Supplementary Figure S2: Epigenetic landscape of the proximal promoter of GFPT2 confirms the location of the NF-B enhancer within the body of the gene locus 127 Supplementary Figure S3: GFPT2 knock-down does not affect EMT in NSCLC 129 Supplementary Figure S4: siRNA-mediated silencing of GFPT2 reduces migration of H1299 NSCLC cells 131 Supplementary Figure S5: O-GlcNAcylation of p65 is elevated in mesenchymal NSCLC cells 133 Figure 7: MEK inhibition activates Akt and NF-B pathways in KRAS-mutant NSCLC cells 142 Figure 8: NF-B activation is required for the survival of KRAS-mutant NSCLC cells following MEK inhibition 145 viii Figure 9: MEK inhibition induces protein O-GlcNAcylation and GFPT2 expression 148 Figure 10: GFPT2 potentiates NF-B signaling in vitro 151 Figure 11: GFPT2 is required for the survival of KRAS-mutant NSCLC cells following MEK inhibition 155 Figure 12: Mesenchymal NSCLC upregulate genes involved in the synthesis of glycans and show elevated multi-branched N-glycosylation 192 Figure 13: GFPT2 interacts with components of the migratory and endocytic machinery 194 ix LIST OF ABBREVIATIONS ADC - adenocarcinoma CIC – cancer initiating cells ECM – extracellular matrix EMT – epithelial to mesenchymal transition GAG - glycosaminoglycan GAPDH - glyceraldehyde-3-phosphate dehydrogenase GFPT – glutamine fructose-6-phosphate aminotransferase HA - hyaluronan HAS – hyaluronan synthase HBP – hexosamine biosynthesis pathway HIF1a – hypoxia-inducible factor 1-alpha IKK – IB kinase NF-kB – nuclear factor kappa B NSCLC – non-small cell lung cancer OGA – O-GlcNAcase OGT – O-linked N-acetylglucosamine (GlcNAc) transferase PG - proteoglycan SCC – squamous cell carcinoma SCLC – small cell lung cancer